48 MORPHOLOGICAL ANOMALIES OF FETAL MEMBRANES DURING GESTATION IN BOVINE CLONES DERIVED BY SOMATIC NUCLEAR TRANSFER

Abstract

High pregnancy loss and prenatal morbidity and mortality encountered in cloned animals might be explained by placental nutritional or steroidogenic insufficiency that would indirectly compromise fetal survival (Hashizume et al. 2002 Cloning and Stem Cells 4(3), 197-209). Our hypothesis is that ultrasonographic characterization of placenta in bovine clones could be used to predict anomalies in fetal development and well-being during pregnancy. We examined surrogate heifers bearing cloned fetuses (n = 37) and fetuses obtained by traditional embryo transfer (n = 5) in three stages of gestation (Day 70, 100 and 200). Morphological parameters based on direct ultrasonographic observations were collected: thickness and shape of amniotic membrane, utero-placental thickness, placentomes shape and length in fetal area, and umbilical cord shape and appearance. Analysis was done using repeated measures linear model, with day as a repeated factor. Of all clone pregnancies, 30 females were still pregnant at Day 70 and only 16 reached Day 100, from which 63% had different degrees of amniotic and/or umbilical cord anomalies. No placental anomalies or abortions were observed in the control group. The amniotic membrane abnormalities observed at Day 100 were irregularities and presence of nodules (rosary-like) in the cross-sectional view of the membrane. Hyperechogenic spikes or irregularities around the umbilical cord were observed at Day 100 in 38% of the clones (n = 6) and all of them also had amniotic anomalies. From the clone pregnancies with amniotic and/or umbilical cord problems, 30% aborted between Days 100 and 200, and for those that went to term (Days 264-278; n = 7), all calves but one died at birth or during the first month of life. Statistical analysis results showed that placentomes in cloned embryos were larger than those in control embryos (P = 0.003; least-squares mean (LSM) for clone = 4.34 cm, control = 2.97 cm) in all three stages of gestation. During the same period, amniotic membranes of clones became thicker compared to those of the control group (P = 0.0001; LSM for clone = 0.25 cm, control = 0.11 cm). Variation in utero-placental thickness was also observed. At Day 70, the utero-placental layer was thicker in clones in comparison to controls (LSM for clone = 0.32 cm, control = 0.30 cm) before it became thinner at Day 200 (LSM for clone = 0.38 cm, control = 0.40 cm). The present results showed that morphological anomalies of placentomes, amniotic membrane and umbilical cord appear during gestation of bovine cloned fetuses that could potentially compromise normal fetal development. Also, ultrasonographic monitoring of pregnancies of cloned animals is a useful tool to characterize the fetal membrane changes.

This work was funded by a Strategic Project Grant from the Natural Sciences and Engineering Research Council (NSERC) Canada.